Programmed cell death-1 (PD-1), encoded by the Pdcd1 gene, is an immune inhibitory receptor that is expressed transiently on the surface of T cells following immune activation but is highly expressed on T cells chronically exposed to antigen. Sustained signaling through PD-1 results in T cell exhaustion, a state in which PD-1 expression is maintained at a high level and where the T cells can no longer respond normally to antigenic challenge. Antibody mediated ?checkpoint? blockade of the PD-1?PDL1/2 pathways in numerous cancers, which results in reinvigoration of T cell immune responses, has had astonishing success in the clinic. PD-1 has been extensively studied in mouse model systems; yet despite its clear therapeutic importance, nearly nothing is known about how PD-1 is regulated in humans! No systematic examination of the cis or trans-regulatory elements of the human PD-1 gene has been conducted and the role of epigenetic pathways regulating its expression is limited. Additionally, PD-1 is highly expressed on TFH cells, yet only limited information regarding its regulation in this cell type is known. Here we seek to fill this knowledge gap by determining how PD-1 is regulated in human CD4, CD8, and TFH T cells. Elucidating the molecular and epigenetic programs that control hPdcd1 expression will provide new understanding, tools, and targets to manipulate PD-1 gene expression that could potentially be used to treat infection, autoimmunity, and cancer. It is clear from the reported data that PD- 1 gene regulation is complex and dependent on the cell type and immune environment/challenge. Therefore, we have focused this application on elucidating the basic transcriptional and epigenetic regulatory mechanisms that control hPdcd1 expression in human T cells. Aim 1 will identify and determine the function of the hPdcd1?s cis-regulatory elements as these are the key to all else that follows. For these experiments, we will use deidentified, peripheral primary human nave, ex vivo activated, and memory CD8 and CD4 T cells, TFH cells, as well as Jurkat cells as a model T cell line. Antigen-specific CD4 and CD8 T cells will be isolated from deidentified, HIV-infected individuals to represent chronically exposed/exhausted T cells. We will integrate epigenomic data (ATAC-seq, ChIP-seq, and bisulfite sequencing) of the above cells to probe the mechanisms by which the cis elements are functioning. Aim 2 will define the transcription factors (TFs) responsible for hPdcd1 regulation. Here we will use genomic information to identify motifs for putative factors and use CRISPR/Cas9 and lentiviral expression systems in primary human T cells to knockout or exogenously express factors and determine their role in regulating hPdcd1. We will use ChIP to define factor binding/occupancy. Lastly, we will use chromatin conformation capture methodologies to demonstrate direct interactions between TFs and their binding sites with the hPdcd1 promoter region and identified enhancers. Together this program will identify the fundamental mechanisms by which hPdcd1 is regulated and will ultimately provide evidence supporting therapies aimed at controlling this pathway through the manipulation of gene regulation.